Methods for identifying immunobinders of cell-surface antigens

ABSTRACT

The invention provides methods for identifying immunobinders, such as scFv antibodies, capable of specifically binding to cell surface antigens, and compositions identified according to said methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. ProvisionalApplication Ser. No. 61/155,041 filed Feb. 24, 2009; U.S. ProvisionalApplication Ser. No. 61/155,105 filed Feb. 24, 2009; Swiss PatentApplication Serial No.: CH/00832/09 filed Jun. 2, 2009; and PCTApplication Serial No.: PCT/CH2009/000222 filed Jun. 25, 20009, theentire contents of which are incorporated herein by reference.

BACKGROUND INFORMATION

Immunobinders, including antibodies, their conjugates and derivativesare hugely commercially important as therapeutic and diagnostic agents.Traditional methods for antibody preparation or screening usuallyutilize soluble antigens. However, for certain membrane-bound proteinantigens, the conformational epitopes on the antigens are altered if theantigens are solubilized from the membrane, resulting in the failure ofantibody preparation or screening. In addition, one major problem inimmunoblotting and affinity chromatography methods is that antibodieswith a moderate affinity for the antigen will be selected. This allowsthe inclusion of many cross-reactive or sticky antibodies, causingburdens in the sequential screening procedures. Although cellsexpressing membrane-bound antigens have been used directly for antibodypreparation, an efficient screening method capable of detecting andenriching high affinity antibodies against cell-surface antigens isstill lacking.

SUMMARY OF THE INVENTION

The invention provides methods for identifying immunobinders, such asscFv antibodies, capable of specifically binding to cell surfaceantigens. The methods of the invention generally comprise contactinglabeled antigen-expressing cells with labeled immunobinder-expressingcells and isolating immunobinder-expressing cells that bind to theantigen-expressing cells using a cell sorter. These methods areparticularly useful for the rapid and efficient identification ofimmunobinders against conformational epitopes present in integralmembrane proteins, such as GPCRs. The invention also provides isolatedimmunobinders and immunobinder-encoding nucleic acids identified usingthe methods of the invention.

In one aspect the invention provides a method for identifying animmunobinder that specifically binds to a cell surface antigen ofinterest. The method comprises: providing a plurality ofimmunobinder-expressing cells operably linked a first sortable label;providing a plurality of antigen-expressing cells operably linked to asecond sortable label, wherein the antigen of interest is displayed atthe surface of the antigen expressing cell; contacting theantigen-expressing cells with the immunobinder-expressing cells; andseparating from the plurality of immunobinder-expressing cells, one ormore immunobinder-expressing cells that can specifically bind to theantigen-expressing cells using a cell sorter (e.g., a fluorescenceactivated cell sorter), wherein the presence of the first and secondsortable label in a single cellular complex (e.g., a complex formedbetween an antigen and a B-cell receptor) is indicative of the bindingof an immunobinder-expressing cell to an antigen-expressing cell,thereby identifying an immunobinder that binds to a antigen of interest.

In some embodiments, the separated immunobinder-expressing cells areclonally isolated. In some embodiments, the immunobinder-expressingcells are subjected to clonal expansion. In other embodiments, theimmunobinder-encoding nucleic acid sequence is isolated fromimmunobinder-expressing cells. Suitable methods for isolation of theimmunobinder-encoding nucleic acid sequence include PCR, e.g.,single-cell PCR. The immunobinder-encoding nucleic acid sequence can beisolated after the cells are clonally isolated and/or after clonalexpansion.

In some embodiments, immunobinder-expressing cells isolated using themethods of the invention are subjected to a cell-based assays in orderto functionally characterize the immunobinder. Suitable cell-basedassays include CELISA.

In some embodiments, the immunobinder is an antibody. Such antibodiesinclude, mouse, rabbit, rabbitized, chicken, camel, camelized, human,humanized and chimeric antibodies. Suitable antibody formats include,without limitation, Fab, Dab, Nanobody and scFv.

In some embodiments, the antigen of interest is expressed from anexogenous gene. In other embodiments, the antigen of interest is agenetically engineered antigen. In other embodiments, the antigen ofinterest is an integral membrane protein. Suitable integral membraneproteins include, without limitation, GPCRs (e.g., CXCR2) or ionchannels.

In some embodiments, the first or second sortable label is a fluorescentlabel. Suitable fluorescent labels include, without limitation,fluorescent proteins, antibody/fluor conjugates and fluorescent cellularlabels.

In some embodiments, the antigen-expressing cells are yeast or mammaliancells (e.g., human cells). In some embodiments, the antigen-expressingcells express an exogenous antigen. In some embodiments theantigen-expressing cells are transfected with an expression vector.

In some embodiments, the immunobinder-expressing cells are yeast ormammalian cells. Suitable mammalian cells include, without limitation,B-cells, e.g., rabbit B-cells. In some embodiments the B-cells areisolated from an immunized animal, e.g., an animal immunized by DNAvaccination. In some embodiments, the immunobinder-expressing cellscomprise an immunobinder expressed from an expression vector.

In another aspect, the invention provides an isolated nucleic acidmolecule encoding an immunobinder identified by the methods of theinvention.

In another aspect, the invention provides a method of producing animmunobinder capable of binding to an antigen of interest, comprisingintroducing an immunobinder-encoding nucleic acid sequence identified bythe methods of the invention into an expression environment such thatthe encoded immunobinder is produced.

In another aspect, the invention provides an immunobinder produced bythe methods of the invention.

In another aspect, the invention also provides a method for identifyinga B-cell clone that specifically binds to a cell surface antigen ofinterest comprising: immunizing an animal with DNA encoding a cellsurface antigen; isolating B-cells from the immunized animal; labelingthe B-cells with a first sortable label; providing a plurality ofantigen-expressing cells operably linked to a second sortable label,wherein the antigen of interest is displayed at the surface of theantigen expressing cell; contacting the antigen-expressing cells withthe B-cells; and separating from the plurality of B-cells, one or moreB-cells that can specifically bind to the antigen-expressing cells usinga cell sorter, wherein the presence of the first and second sortablelabel in a single cellular complex is indicative of the binding of anB-cell to an antigen-expressing cell, thereby identifying a B-cell clonethat binds to a antigen of interest.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically shows a B-cell 1 labeled with a fluorescentantibody 2 interacting with a target-expressing cell stained with anintracellular dye 3. (4: target of choice/antigen; 5: B cell receptor(BCR)).

FIGS. 2A-2C: the FACS selection process of rabbit B cells binding toESBA903 soluble target. FIG. 2A: Lymphocytes were gated according toforward and side scatter. FIG. 2B: Among them, IgG+ IgM− cells (probablymemory B cells) were selected (shown circled). FIG. 2C: Cellsdouble-stained with ESBA903-PE and ESBA903-PerCP (shown circled) wereexpected to encode high affinity IgGs against ESBA903. Cells showing thebrightest fluorescence (uncircled) were sorted in 96-well plates.

FIG. 3: Beads coated with anti-TNFalpha antibodies (PE labeled) bind toTNFalpha-transfected CHO cells (upper panel). Control beads coatedanti-CD19 antibodies (APC labeled) did not bind TNFalpha transfected CHOcells (middle panel). Beads coated with anti-TNFalpha antibodies (PElabeled) did not bind to wildtype (wt) CHO cells (lower panel). Dotplots on the left show forward and side scatters, which indicaterespectively the size and the granularity of the events. The singlebeads (˜3 um) population was gated in P2. CHO cells eventually bound tobeads (˜30 um) were gated in P1. Dot plots in the middle show the P1events (CHO cells) in respect to their PE or APC staining Thus, cellsinteracting with anti-TNFalpha beads would appear in P3 gate, and cellsinteracting with the anti-CD19 beads would appear in P4 gate. On theright, statistics for each sample are detailed.

FIG. 4: Beads coated with anti-TNFalpha-PE and beads coated withanti-CD19-APC were mixed together with TNFalpha-transfected CHO cells.CHO cells were gated (P1), and among them cells binding to eitheranti-TNFalphaPE coated beads or anti-CD19-APC coated beads are shown ingates P3 and P4, respectively. Unbound beads are visible in gate P2.

FIGS. 5A-5B: FIG. 5 a: FACS analysis of the 3 different CIO-TNFα—memoryB cells suspensions. Top left: dot plot showing cell suspension forwardand side scatter. The living cells, comprising a large population oftransgenic CHO cells and a small population of memory B cells, weregated. Down left: dot plot showing APC and FITC fluorescence. Here, thememory B cells (IgG+/IgM−) were gated. These two dot plots wereidentical for all three samples; therefore, they are shown only once.

FIG. 5 b: histograms and population hierarchy of the 3 samples: top:CHO-TNFα cells+ESBA105+memory B cells of ESBA105 immunized rabbit;middle: CHO-TNFα cells+ESBA105+memory B cells of non-immunized rabbit;down: CHO-TNFα cells+memory B cells of ESBA105 immunized rabbit. On thehistograms, memory B cells binding to CHO cells were gated.

FIGS. 6A-6C: FACS analysis of the suspension consisting of immunizedlymphocytes mixed with TNFα transgenic CHO cells “coated” with ESBA105.FIG. 6 a: dot plot showing cell suspension forward and side scatter. Theliving cells, comprising a large population of transgenic CHO cells anda small population of lymphocytes, were gated. FIG. 6 b: dot plotshowing APC and FITC fluorescence. Here, the memory B cells (IgG+/IgM−)were gated. FIG. 6 c: histogram showing calcein fluorescence of gatedmemory B cells. Gated population was sorted (memory B cells binding toCHO-TNFα-ESBA105 complex).

FIG. 7: Bright field microscopy pictures of beads coated with anti-TNFαIgG which interact with CHO-TNFα (B220) transgenic cells.

FIGS. 8A-8B: Bright Field microscopy pictures (left column: FIG. 8 a)and fluorescence microscopy pictures (right column: FIG. 8 b) ofCHO-TNFalpha/ESBA105 cells (big cells) bound to B cells, which haveanti-ESBA105 antibodies on the surface (smaller cells).

DETAILED DESCRIPTION

The invention provides methods for identifying immunobinders, such asscFv antibodies, capable of specifically binding to cell surfaceantigens. The methods of the invention generally comprise contactinglabeled antigen-expressing cells with labeled immunobinder-expressingcells and isolating immunobinder-expressing cells that bind to theantigen-expressing cells using a cell sorter. These methods areparticularly useful for the rapid and efficient identification ofimmunobinders against conformational epitopes present in integralmembrane proteins, such as GPCRs. The invention also provides isolatedimmunobinders and immunobinder-encoding nucleic acids identified usingthe methods of the invention.

In one aspect the invention provides a method for identifying animmunobinder that specifically binds to a cell surface antigen ofinterest. The method comprises: providing a plurality ofimmunobinder-expressing cells operably linked to a first sortable label;providing a plurality of antigen-expressing cells operably linked to asecond sortable label, wherein the antigen of interest is displayed atthe surface of the antigen expressing cell; contacting theantigen-expressing cells with the immunobinder-expressing cells; andseparating from the plurality of immunobinder-expressing cells, one ormore immunobinder-expressing cells that can specifically bind to theantigen-expressing cells using a cell sorter (e.g., a fluorescenceactivated cell sorter), wherein the presence of the first and secondsortable label in a single cellular complex (e.g., a complex formedbetween an antigen and a B-cell receptor) is indicative of the bindingof an immunobinder-expressing cell to an antigen-expressing cell,thereby identifying an immunobinder that binds to an antigen ofinterest.

In some embodiments, the separated immunobinder-expressing cells areclonally isolated.

In certain embodiments, the clonally isolated immunobinder-expressingcells are subjected to clonal expansion using methods well-known tothose of skill in the art.

In other embodiments, the immunobinder-encoding nucleic acid sequence isisolated from immunobinder-expressing cells. Isolation of the nucleicacid sequence can occur after clonal isolation or after clonalexpansion. Suitable methods for isolation of the immunobinder-encodingnucleic acid sequence include PCR, e.g., single-cell PCR.

In some embodiments, immunobinder-expressing cells isolated using themethods of the invention are subject to a cell-based assays in order tofunctionally characterize the immunobinder. Suitable cell-based assaysinclude CELISA.

In some embodiments, the immunobinder is an antibody. Such antibodiesinclude, mouse, rabbit, rabbitized, chicken, camel, camelized, human,humanized and chimeric antibodies. Suitable antibody formats include,without limitation, Fab, Dab, Nanobody and scFv.

In some embodiments, the antigen of interest is expressed from anexogenous gene. In other embodiments, the antigen of interest is agenetically engineered antigen. In other embodiments, the antigen ofinterest is an integral membrane protein. Suitable integral membraneproteins include, without limitation, G protein-coupled receptors(GPCRs, such as CXCR2) or ion channels.

In some embodiments, the first or second sortable label is a fluorescentlabel. Suitable fluorescent labels include, without limitation,fluorescent proteins, antibody/fluor conjugates and fluorescent cellularlabels.

In some embodiments, the antigen-expressing cells are yeast or mammaliancells (e.g., human cells). In some embodiments, the antigen-expressingcells express an exogenous antigen. In some embodiments theantigen-expressing cells are transfected with an expression vector.

In some embodiments, the immunobinder-expressing cells are yeast ormammalian cells. Suitable mammalian cells include, without limitation,B-cells, e.g., rabbit B-cells. In some embodiments the B-cells areisolated from an immunized animal, e.g., an animal immunized by DNAvaccination. In some embodiments, the immunobinder-expressing cellscomprise an immunobinder expressed from an expression vector.

In another aspect, the invention provides an isolated nucleic acidmolecule encoding an immunobinder identified by the methods of theinvention.

In another aspect, the invention provides a method of producing animmunobinder capable of binding to an antigen of interest, comprisingintroducing an immunobinder-encoding nucleic acid sequence identified bythe methods of the invention into an expression environment such thatthe encoded immunobinder is produced.

In another aspect, the invention provides an immunobinder produced bythe methods of the invention.

In another aspect, the invention also provides a method for identifyinga B-cell clone that specifically binds to a cell surface antigen ofinterest comprising: immunizing an animal with DNA encoding a cellsurface antigen; isolating B-cells from the immunized animal; labelingthe B-cells with a first sortable label; providing a plurality ofantigen-expressing cells operably linked to a second sortable label,wherein the antigen of interest is displayed at the surface of theantigen expressing cell; contacting the antigen-expressing cells withthe B-cells; and separating from the plurality of B-cells, one or moreB-cells that can specifically bind to the antigen-expressing cells usinga cell sorter, wherein the presence of the first and second sortablelabel in a single cellular complex is indicative of the binding of anB-cell to an antigen-expressing cell, thereby identifying a B-cell clonethat binds to a antigen of interest.

DEFINITIONS

In order that the present invention may be more readily understood,certain terms will be defined as follows. Additional definitions are setforth throughout the detailed description.

The term “antibody” refers to whole antibodies and any antigen bindingfragment (i.e., “antigen-binding portion,” “antigen bindingpolypeptide,” or “immunobinder”) or single chain thereof. An “antibody”refers to a glycoprotein comprising at least two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, or an antigenbinding portion thereof. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as VH) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (abbreviated herein as VL) and a light chain constantregion. The light chain constant region is comprised of one domain, CL.The VH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

The term “chimeric antibody” refers to an antibody molecule in which (a)the constant region, or a portion thereof, is altered, replaced orexchanged so that the antigen binding site (variable region) is linkedto a constant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”) refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., TNF). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a single domain such as a dAb fragment (Ward et al.,(1989) Nature 341:544-546), which consists of a VH domain; and (vi) anisolated complementarity determining region (CDR) or (vii) a combinationof two or more isolated CDRs which may optionally be joined by asynthetic linker. Furthermore, although the two domains of the Fvfragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies. Antigen-binding portions can be produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intactimmunoglobulins. Antibodies can be of different isotype, for example, anIgG (e.g., an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE,or IgM antibody.

The term “immunobinder” refers to a molecule that contains all or a partof the antigen binding site of an antibody, e.g., all or part of theheavy and/or light chain variable domain, such that the immunobinderspecifically recognizes a target antigen. Non-limiting examples ofimmunobinders include full-length immunoglobulin molecules and scFvs, aswell as antibody fragments, including but not limited to (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fab′fragment, which is essentially a Fab with part of the hinge region (see,FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed. 1993); (iv) a Fd fragmentconsisting of the VH and CH1 domains; (v) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (vi) a singledomain antibody such as a Dab fragment (Ward et al., (1989) Nature341:544-546), which consists of a VH or VL domain, a Camelid (seeHamers-Casterman, et al., Nature 363:446-448 (1993), and Dumoulin, etal., Protein Science 11:500-515 (2002)) or a Shark antibody (e.g., sharkIg-NARs Nanobodies®; and (vii) a nanobody, a heavy chain variable regioncontaining a single variable domain and two constant domains.

As used herein, the term “functional property” is a property of apolypeptide (e.g., an immunobinder) for which an improvement (e.g.,relative to a conventional polypeptide) is desirable and/or advantageousto one of skill in the art, e.g., in order to improve the manufacturingproperties or therapeutic efficacy of the polypeptide. In oneembodiment, the functional property is improved stability (e.g., thermalstability). In another embodiment, the functional property is improvedsolubility (e.g., under cellular conditions). In yet another embodiment,the functional property is non-aggregation. In still another embodiment,the functional property is an improvement in expression (e.g., in aprokaryotic cell). In yet another embodiment the functional property isan improvement in refolding yield following an inclusion bodypurification process. In certain embodiments, the functional property isnot an improvement in antigen binding affinity.

The term “frameworks” refers to the art recognized portions of anantibody variable region that exist between the more divergent CDRregions. Such framework regions are typically referred to as frameworks1 through 4 (FR1, FR2, FR3, and FR4) and provide a scaffold for holding,in three-dimensional space, the three CDRs found in a heavy or lightchain antibody variable region, such that the CDRs can form anantigen-binding surface. Such frameworks can also be referred to asscaffolds as they provide support for the presentation of the moredivergent CDRs. Other CDRs and frameworks of the immunoglobulinsuperfamily, such as ankyrin repeats and fibronectin, can be used asantigen binding molecules (see also, for example, U.S. Pat. Nos.6,300,064, 6,815,540 and U.S. Pub. No. 20040132028).

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds. Anepitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15 amino acids in a unique spatial conformation. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

The terms “specific binding”, “selective binding”, “selectively binds”,and “specifically binds” refer to antibody binding to an epitope on apredetermined antigen. Typically, the antibody binds with an affinity(K) of approximately less than 10⁻⁷ M, such as approximately less than10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower.

The term “K_(D),” refers to the dissociation equilibrium constant of aparticular antibody-antigen interaction. Typically, the antibodies ofthe invention bind to an antigen with a dissociation equilibriumconstant (K_(D)) of less than approximately 10-7 M, such as less thanapproximately 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower, for example, asdetermined using surface plasmon resonance (SPR) technology in a BIACOREinstrument.

As used herein, “identity” refers to the sequence matching between twopolypeptides, molecules or between two nucleic acids. When a position inboth of the two compared sequences is occupied by the same base or aminoacid monomer subunit (for instance, if a position in each of the two DNAmolecules is occupied by adenine, or a position in each of twopolypeptides is occupied by a lysine), then the respective molecules areidentical at that position. The “percentage identity” between twosequences is a function of the number of matching positions shared bythe two sequences divided by the number of positions compared×100. Forinstance, if 6 of 10 of the positions in two sequences are matched, thenthe two sequences have 60% identity. By way of example, the DNAsequences CTGACT and CAGGTT share 50% identity (3 of the 6 totalpositions are matched). Generally, a comparison is made when twosequences are aligned to give maximum identity. Such alignment can beprovided using, for instance, the method of Needleman et al. (1970) J.Mol. Biol. 48: 443-453, implemented conveniently by computer programssuch as the Align program (DNAstar, Inc.). The percent identity betweentwo amino acid sequences can also be determined using the algorithm ofE. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) whichhas been incorporated into the ALIGN program (version 2.0), using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. In addition, the percent identity between two amino acidsequences can be determined using the Needleman and Wunsch (J. Mol.Biol. 48:444-453 (1970)) algorithm which has been incorporated into theGAP program in the GCG software package (available at www.gcg.com),using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

“Similar” sequences are those which, when aligned, share identical andsimilar amino acid residues, where similar residues are conservativesubstitutions for corresponding amino acid residues in an alignedreference sequence. In this regard, a “conservative substitution” of aresidue in a reference sequence is a substitution by a residue that isphysically or functionally similar to the corresponding referenceresidue, e.g., that has a similar size, shape, electric charge, chemicalproperties, including the ability to form covalent or hydrogen bonds, orthe like. Thus, a “conservative substitution modified” sequence is onethat differs from a reference sequence or a wild-type sequence in thatone or more conservative substitutions are present. The “percentagesimilarity” between two sequences is a function of the number ofpositions that contain matching residues or conservative substitutionsshared by the two sequences divided by the number of positionscompared×100. For instance, if 6 of 10 of the positions in two sequencesare matched and 2 of 10 positions contain conservative substitutions,then the two sequences have 80% positive similarity.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not negativelyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative sequence modificationsinclude nucleotide and amino acid substitutions, additions anddeletions. For example, modifications can be introduced by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative amino acid substitutions includeones in which the amino acid residue is replaced with an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Thus, a predicted nonessential amino acidresidue may be replaced with another amino acid residue from the sameside chain family. Methods of identifying nucleotide and amino acidconservative substitutions which do not eliminate antigen binding arewell-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187(1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burkset al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

“Amino acid consensus sequence” as used herein refers to an amino acidsequence that can be generated using a matrix of at least two, andpreferably more, aligned amino acid sequences, and allowing for gaps inthe alignment, such that it is possible to determine the most frequentamino acid residue at each position. The consensus sequence is thatsequence which comprises the amino acids which are most frequentlyrepresented at each position. In the event that two or more amino acidsare equally represented at a single position, the consensus sequenceincludes both or all of those amino acids.

The amino acid sequence of a protein can be analyzed at various levels.For example, conservation or variability can be exhibited at the singleresidue level, multiple residue level, multiple residue with gaps, etc.Residues can exhibit conservation of the identical residue or can beconserved at the class level. Examples of amino acid classes includepolar but uncharged R groups (Serine, Threonine, Asparagine andGlutamine); positively charged R groups (Lysine, Arginine, andHistidine); negatively charged R groups (Glutamic acid and Asparticacid); hydrophobic R groups (Alanine, Isoleucine, Leucine, Methionine,Phenylalanine, Tryptophan, Valine and Tyrosine); and special amino acids(Cysteine, Glycine and Proline). Other classes are known to one of skillin the art and may be defined using structural determinations or otherdata to assess substitutability. In that sense, a substitutable aminoacid can refer to any amino acid which can be substituted and maintainfunctional conservation at that position.

It will be recognized, however, that amino acids of the same class mayvary in degree by their biophysical properties. For example, it will berecognized that certain hydrophobic R groups (e.g., Alanine,) are morehydrophilic (i.e., of higher hydrophilicity or lower hydrophobicity)than other hydrophobic R groups (e.g., Valine or Leucine). Relativehydrophilicity or hydrophobicity can be determined using art-recognizedmethods (see, e.g., Rose et al., Science, 229: 834-838 (1985) andCorvette et al., J. Mol. Biol., 195: 659-685 (1987)).

As used herein, when one amino acid sequence (e.g., a first VH or VLsequence) is aligned with one or more additional amino acid sequences(e.g., one or more VH or VL sequences in a database), an amino acidposition in one sequence (e.g., the first VH or VL sequence) can becompared to a “corresponding position” in the one or more additionalamino acid sequences. As used herein, the “corresponding position”represents the equivalent position in the sequence(s) being comparedwhen the sequences are optimally aligned, i.e., when the sequences arealigned to achieve the highest percent identity or percent similarity.

The term “nucleic acid molecule,” refers to DNA molecules and RNAmolecules. A nucleic acid molecule may be single-stranded ordouble-stranded, but preferably is double-stranded DNA. A nucleic acidis “operably linked” when it is placed into a functional relationshipwith another nucleic acid sequence. For instance, a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence.

The term “vector,” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid,” which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.

The term “host cell” refers to a cell into which an expression vectorhas been introduced. Host cells can include bacterial, microbial, plantor animal cells. Bacteria, which are susceptible to transformation,include members of the enterobacteriaceae, such as strains ofEscherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis;Pneumococcus; Streptococcus, and Haemophilus influenzae. Suitablemicrobes include Saccharomyces cerevisiae and Pichia pastoris. Suitableanimal host cell lines include CHO (Chinese Hamster Ovary lines) and NS0cells.

The terms “treat,” “treating,” and “treatment,” refer to therapeutic orpreventative measures described herein. The methods of “treatment”employ administration to a subject, in need of such treatment, anantibody of the present invention, for example, a subject having aGPCR-mediated disorder or a subject who ultimately may acquire such adisorder, in order to prevent, cure, delay, reduce the severity of, orameliorate one or more symptoms of the disorder or recurring disorder,or in order to prolong the survival of a subject beyond that expected inthe absence of such treatment.

The term “effective dose” or “effective dosage” refers to an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountseffective for this use will depend upon the severity of the disorderbeing treated and the general state of the patient's own immune system.

The term “subject” refers to any human or non-human animal. For example,the methods and compositions of the present invention can be used totreat a subject with a GPCR-mediated disorder.

The term “rabbit” as used herein refers to an animal belonging to thefamily of the leporidae.

The term “cell sorter” refers to any means for separating cells basedupon the presence of a detectable “sortable” label. Such means include,without limitation, a fluorescence activation cell sorter. Any cellularlabels can be used as sortable labels including, without limitation,fluorescent proteins, e.g. Green fluorescent protein, antibody/fluorconjugates and fluorescent cellular labels, e.g., fluorescent calciumionophores

The term “cellular complex” refers to a one or more antigen-expressingcells bound to one or more immunobinder-expressing cells, wherein thebinding is mediated by the antigen on the surface of theantigen-expressing cell. In some embodiments, the binding of anantigen-expressing cell to an immunobinder-expressing cell consists of adirect interaction between the antigen on the surface of theantigen-expressing cell and the immunobinder on the surface of theimmunobinder-expressing cell.

The term “clonally isolating” refers to any means for the isolation ofindividual cell clones from a cell population. Suitable means include,without limitation, the limiting dilution and transfer of cells tomultiwell plates such that each well contains no more than a singlecell.

The term “obtaining the immunobinder-encoding nucleic acid sequence”refers to any means for obtaining the nucleic acid sequence of animmunobinder expressed by an immunobinder-expressing cell. Suitablemeans include, without limitation, nucleic acid isolation, PCRamplification and DNA sequencing of the immunobinder-encoding nucleicacid sequence from the immunobinder-expressing cell. In someembodiments, immunobinder-encoding nucleic acid sequences are amplifiedby PCR from single cells, i.e. “single cell PCR”.

The term “exogenous antigen” refers to an antigen not normally expressedin a particular host cell. For example, an exogenous antigen can be froma different kingdom, phylum class, order, genus or species from the hostcell, e.g., a human antigen expressed in a yeast cell. Additionally oralternatively, an exogenous antigen can be from the same species butinappropriately expressed in that host cell, e.g, a lung specificantigen expressed in a brain cell. “Exogenous antigen” also refers tomutant an antigen not normally found in a normal cell, e.g., a cancerspecific mutant antigen expressed in a lung cell.

The term “genetically engineered antigen” refers to any antigen that hasbeen produced by recombination DNA techniques and includes antigens thatare chimeras or contain point mutation, deletions and/or insertions.Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Various aspects of the invention are described in further detail in thefollowing subsections. It is understood that the various embodiments,preferences and ranges may be combined at will. Further, depending ofthe specific embodiment, selected definitions, embodiments or ranges maynot apply.

The present invention provides a screening method using FACS to identifyand separate immunobinder-expressing cells in adherence with cellsexpressing corresponding antigen. In particular embodiments, theimmunobinder is an antibody.

Antigen Expression

The target antigen for antibody preparation can be any protein, peptide,nucleotide, carbohydrate, lipid, and other molecules that are soluble orexpressed on a cell surface or integrated in the plasma membrane.Antigens can be native or synthetic. Preferably, a target antigen is aprotein or peptide. Non-limiting examples of a target antigen includeCXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6,CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC-Kb,Bestrophins, TMEM16A, GABA receptor, glycin receptor, ABC transporters,NAV1.1, NAV1.2, NAV1.3, NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9,sphingosin-1-phosphate receptor (S1P1R), NMDA channel etc. In oneembodiment, the target antigen is a transmembrane protein. In anotherembodiment, the target antigen is a multispan transmembrane protein, forexample, G protein coupled receptors (GPCRs), ion channels, etc.

The family of GPCRs has at least 250 members (Strader et al. FASEB J.,9:745-754, 1995; Strader et al. Annu Rev. Biochem., 63:101-32, 1994). Ithas been estimated that one percent of human genes may encode GPCRs.GPCRs bind to a wide-variety of ligands ranging from photons, smallbiogenic amines (i.e., epinephrine and histamine), peptides (i.e.,IL-8), to large glycoprotein hormones (i.e., parathyroid hormone). Uponligand binding, GPCRs regulate intracellular signaling pathways byactivating guanine nucleotide-binding proteins (G proteins).Interestingly, GPCRs have functional homologues in human cytomegalovirusand herpesvirus, suggesting that GPCRs may have been acquired duringevolution for viral pathogenesis (Strader et al., FASEB J., 9:745-754,1995; Arvanitakis et al. Nature, 385:347-350, 1997; Murphy, Annu Rev.Immunol. 12:593-633, 1994).

The characteristic feature of most GPCRs which have been known up to nowis that seven clusters of hydrophobic amino acid residues are located inthe primary structure and pass through (span) the cell membrane at eachregion thereof. The domains are believed to represent transmembranealpha-helices connected by three intracellular loops, threeextracellular loops, and amino- and carboxyl-terminal domains (K.Palczewski et al., Science 289, 739-45 (2000)). Most GPCRs have singleconserved cysteine residues in each of the first two extracellular loopswhich form disulfide bonds that are believed to stabilize functionalprotein structure. The 7 transmembrane regions are designated as TM1,TM2, TM3, TM4, TM5, TM6, and TM7. It is well known that these structuresdetailed above are common among G protein coupled receptor proteins andthat the amino acid sequences corresponding to the area where theprotein passes through the membrane (membrane-spanning region ortransmembrane region) and the amino acid sequences near themembrane-spanning region are often highly conserved among the receptors.Thus, due to the high degree of homology in GPCRs, the identification ofnovel GPCRs, as well as identification of both the intracellular and theextracellular portions of such novel members, is readily accomplished bythose of skill in the art. By way of example, the book of Watson andArkinstall (1994), incorporated herein by reference, provides thesequences of over 50 GPCRs. The book further describes, for eachsequence, the precise residues comprising each of the transmembranedomains.

The binding sites for small ligands of G-protein coupled receptors arebelieved to comprise a hydrophilic socket located near the extracellularsurface and formed by several G-protein coupled receptors transmembranedomains, which socket is surrounded by hydrophobic residues of theG-protein coupled receptors. The hydrophilic side of each G-proteincoupled receptor transmembrane helix is postulated to face inward andform the polar ligand binding site. TM3 has been implicated in severalG-protein coupled receptors as having a ligand binding site, such asincluding the TM3 aspartate residue. Additionally, TM5 serines, a TM6asparagine and TM6 or TM7 phenylalanines or tyrosines are alsoimplicated in ligand binding. The ligand binding site for peptidehormones receptors and receptors with other larger ligands such asglycoproteins (LH, FSH, hCG, TSH), and the Ca2+/glutamate/GABA classesof receptors likely residue in the extracellular domains and loops.

A key event for the switch from inactive to active receptor isligand-induced conformational changes of transmembrane helices 3 (TM3)and 6 (TM6) of the GPCRs that have 7 transmembrane spanning helices (U.Gether, and B. K. Kolbilka, J. Biol. Chem. 273, 17979-17982 (1998)).These helical movements in turn alter the conformation of theintracellular loops of the receptor to promote activation of associatedheterotrimeric G proteins. Mutagenesis studies (S. Cotecchia, J.Ostrowski, M. A. Kjelsberg, M. G. Caron and R. J. Lefkowitz, J. Biol.Chem. 267, 1633-1639 (1992); E. Kostenis, B. R. Conklin and J. Wess,Biochemistry 36, 1487-1495 (1997); M. A. Kjelsberg, S. Coteechia, J.Ostrowski, M. G. Caron, and R. J. Lefkowitz, J. Biol. Chem. 267,1430-1433 (1992)) demonstrated that the third intracellular loop (i3)mediates a large part of the coupling between receptor and G protein. I3loops expressed as minigenes have also been shown to directly competewith adrenergic receptors for Gq binding (L. M. Luttrell, J. Ostrowski,S. Cotecchia, H. Kendal and R. J. Lefkowitz, Science 259, 1453-1457(1993)), or can activate G proteins as soluble peptides in cell-freeconditions (T. Okamoto et al., Cell 67, 723-730 (1991)).

The antigen of interest can be of endogenous source in the target cell(sometimes also referred to as antigen-expressing cell). Alternatively,exogenous molecules can be introduced into cells to express the antigen.The introduction of the antigen into cells can be practiced by anymethod known to a person with ordinary skills in the art. In oneembodiment, a polynucleotide encoding the antigen as a polypeptide canbe inserted in vitro into a vector, which can further be introduced intotarget cells for expression. The polynucleotide can contain the cDNAsequence, DNA sequence, or other sequences known in the art, of thetarget antigen. The vector can be plasmid, cosmid, liposome, or othernatural or artificial vectors known in the art. The introduction can bea process of transfection, transformation, infection, directmicro-injection of materials, biolistic particle delivery,electroporation, or other methods known in the art. The target cellexpressing the antigen can be any cells known in the art, including, forexample, cells directly taken from animals, e.g., cancer cells,non-cancer cells, primary cells, etc., or cells with molecularengineering, e.g., culture cells (e.g., Chinese Hamster Ovary (CHO)cells, HEK293 cells, etc.), immortalized cells, transfected/infectedcells, T cells, etc. Alternatively, the target cell can come fromnon-animal sources, e.g., bacterial, insect, etc. In one embodiment, thecells expressing the target antigen are yeast cells, preferably yeastspheroblasts. Alternatively, the target “cell” can be an artificialcell-like body or structure, e.g., liposome, single-layer membrane body,etc. The expression of the antigen in the target cell or cell-likebodies can be transient, i.e., the expression will attenuate or stopafter a comparatively short period of time (e.g., from minutes toseveral days), or stable, i.e., the expression will sustain at acomparatively stable level for a comparatively long time (e.g., afterseveral days or several generations of cells). In one preferableembodiment, the antigen is expressed on the extracellular surface of theplasma membrane of the target cell. In another preferable embodiment,the antigen is an integral or multispan membrane antigen. To get to itslocation on the plasma membrane, the antigen can be expressed directlyon these locations, or can be translocated to these locations aftertheir expression in the cytoplasm of the target cell. This translocationcan be a natural process of the target cell or an engineered process by,for example, attaching a signal/tag molecule (e.g., Golgi sortingsignals, antibodies to certain membrane-bound molecules, etc.),anchoring graft (e.g., a glycosylphosphatidylinositol (GPI) anchor), orchemical crosslink to the antigen, mutating the antigen, or othermethods known in the art, before or after the antigen expression, whichleads to its translocation.

Immunobinder-Expressing Cells and Immunization

In one embodiment, the immunobinder-expressing cells to be selected bythe method described herein are mammalian B cells, preferably rabbitB-cells.

In a preferred embodiment, the B cells originate from an animal that wasimmunized with the target of interest. The immunization of the animalcan be practiced by any method known to a person with ordinary skills inthe art. Typically, the B-cells are isolated from lymphatic organs of animmunized animal (such as spleen or lymph nodes).

In one preferred embodiment, the immunization of antigen of the interestis done by DNA immunization/vaccination. Alternatively, cells expressingthe target antigen are injected into animals (e.g., rabbit, rat, mouse,hamster, sheep, goat, chicken, etc.) for the immunization. The preferredanimal for this immunization step is a rabbit. DNAimmunization/vaccination induces a rapid immune response and allows fornative expression, and usually only native expression, of targetantigens. Because it does not involve the expression and handling ofrecombinant proteins, this process is more efficient and cost-effectivethan traditional immunization with recombinant proteins. In addition,more importantly, the in vivo expressed antigen possesses the samesecondary structures and may even possess the same post-translationalmodifications as the target protein in its natural context, whichimproves the correctness of recognition by the prepared antibodiesagainst the target antigen. An exemplary DNA immunization is illustratedin a Canadian patent application CA2350078 and in WO04/087216.Specifically, the DNA encoding the polypeptide as the target antigen isintroduced directly into an animal through a gene gun method, resultingin expression of a polypeptide in the animal, which expression causesthe formation of antibodies against the polypeptide. In order to achievea more vigorous antibody formation, so-called genetic adjuvants areapplied simultaneously with the polypeptide-encoding DNA. These geneticadjuvants are plasmids which express cytokines (e.g., GM-CSF, IL-4 andIL-10) and which stimulate the humoral immune response in the laboratoryanimals. In one preferred embodiment, the antibody to the target antigenis expressed on the cell surface of B cells as a B cell receptor (BCR).In another preferred embodiment, the antibody-expressing cell is amemory B cell, characterized and distinguishable from regular B cells bythe absence of any IgM on its surface.

In one embodiment, the immunobinder-expressing cells are yeast cells andthe immunobinders are preferably antibody fragments, more preferablyscFv.

Screening Using Fluorescent-Activated Cell Sorting (FACS)

After the immunization step, the membrane-bound antibody on B cells thatspecifically binds to the target antigen need to be separated from othercells expressing non-specific antibodies. In one preferred embodiment,through the antigen-antibody binding, B cells expressing the specificantibody on its plasma membrane is adhered to the target cellsexpressing the antigen. In another one preferred embodiment, other ormore interactions (e.g., same or different antigen-antibodyinteractions, chemical crosslinks, ligand-receptor interactions, etc.)may occur between B cells and target cells. The B cells can be in a poolof B cells expressing different antibodies, or in combination with otherimmune cells, collected directly from the immunized animal, from a poolof immunized/non-immunized animals, or from in vitro engineeringprocesses, for example, a library of B cells expressing differentantibodies by V(D)J genetic recombination technique.

The separation of the B cells expressing antibodies specific to thetarget antigen can be done in any method known in the art. Theseinclude, but are not limited to, panning on antigen, limited dilution,affinity purification, or other methods utilizing the characteristics ofthe expressed antibodies or the antibody-producing B cells.

In one preferred embodiment, the antigen-expressing cells are labeledwith a tag useful for future detection and/or isolation of the adhered Bcells. The tag can be a crosslinker, antigen/antibody, small molecules(e.g., glutathione (GSH), biotin/avidin, etc.), magnetic particles,fluorescence tag, etc. In one preferred embodiment, theantigen-expressing cells are labeled with a fluorescent protein/peptide.In another embodiment, the antibody-producing B cells are also labeledwith a tag, preferably, a different fluorescent protein/peptide. In yetanother embodiment, the B cells in adherence to the antigen-expressingcells can be detected by the emission of fluorescence from thefluorescent protein/peptide labeled on B cells. In still anotherembodiment, the B cell and the antigen-expressing cell in adherence canbe detected by the emission of fluorescence from two differentfluorescent protein/peptide labeled on them. In one preferredembodiment, B cells expressing antibodies specific to the target antigencan be detected and further separated from other antibody-producing Bcells by the emission of both fluorescence from fluorescentproteins/peptides labeled on them and on adhered APCs. Preferably, thisdetection and separation can be performed by fluorescence-activated cellsorting (FACS) technique.

The acronym FACS is trademarked and owned by Becton Dickinson (FranklinLakes, N.J.). As used herein, the term FACS shall represent any form offlow cytometry based cell sorting.

Fluorescence-activated cell sorting is a specialized type of flowcytometry. It provides a method for sorting a heterogeneous mixture ofbiological cells into two or more containers, one cell at a time, basedupon the specific light scattering and fluorescent characteristics ofeach cell. It is a useful scientific instrument as it provides fast,objective and quantitative recording of fluorescent signals fromindividual cells as well as physical separation of cells of particularinterest.

In a typical FACS system, the cell suspension is entrained in the centerof a narrow, rapidly flowing stream of liquid. The flow is arranged sothat there is a large separation between cells relative to theirdiameter. A vibrating mechanism causes the stream of cells to break intoindividual droplets. The system is adjusted so that there is a lowprobability of more than one cell being in a droplet. Just before thestream breaks into droplets the flow passes through a fluorescencemeasuring station where the fluorescent character of interest of eachcell is measured. An electrical charging ring is placed just at thepoint where the stream breaks into droplets. A charge is placed on thering based on the immediately prior fluorescence intensity measurementand the opposite charge is trapped on the droplet as it breaks from thestream. The charged droplets then fall through an electrostaticdeflection system that diverts droplets into containers based upon theircharge. In some systems the charge is applied directly to the stream andthe droplet breaking off retains charge of the same sign as the stream.The stream is then returned to neutral after the droplet breaks off.

The fluorescent labels for FACS technique depend on the lamp or laserused to excite the fluorochromes and on the detectors available. Themost commonly available lasers on single laser machines are blue argonlasers (488 nm). Fluorescent labels workable for this kind of lasersinclude, but not limited to, 1) for green fluorescence (usually labelledFL1): FITC, Alexa Fluor 488, GFP, CFSE, CFDA-SE, and DyLight 488; 2) fororange fluorescence (usually FL2): PE, and PI; 3) for red fluorescence(usually FL3): PerCP, PE-Alexa Fluor 700, PE-Cy5 (TRI-COLOR), andPE-Cy5.5; and 4) for infra-red fluorescence (usually FL4; in some FACSmachines): PE-Alexa Fluor 750, and PE-Cy7. Other lasers and theircorresponding fluorescent labels include, but are not limited to, 1) reddiode lasers (635 nm): Allophycocyanin (APC), APC-Cy7, Alexa Fluor 700,Cy5, and Draq-5; and 2) violet lasers (405 nm): Pacific Orange, AmineAqua, Pacific Blue, 4′,6-diamidino-2-phenylindole (DAPI), and AlexaFluor 405.

In a preferred embodiment, B cells are stained with labeled anti-IgG andanti-IgM antibodies and the memory B cells only being positively stainedwith the anti-IgG antibodies but not with the anti-IgM antibodies arepreferentially selected. IgG have generally a higher affinity than IgM;positive B-cells expressing IgG but not IgM on their surface (which ischaracteristic for memory B-cells) are thereby selected. For saidpurpose, multicolor staining is preferably used, where antibodiesspecific for IgG and IgM are differentially labeled, e.g. with APC andFITC, respectively. Preferably, the target antigen and/or the targetcell expressing the target antigen are also labeled. In one embodiment,the target antigen is stained indirectly by staining the cell thatexpresses the target antigen with an intracellular fluorescent dye.

The present invention provides a method using FACS to screen B cellspools, in which B cells may adhere to cells expressing target antigens,to identify and further isolate B cells producing antibodiesspecifically binding to the target antigen of interest. Preferably, theB cells are labeled with a fluorescent label and the cells expressingthe target antigen are separately labeled with a different fluorescentlabel. These labels can be either intracellular, extracellular, orintegrated in the plasma membrane. After the immunization and productionof antibodies, all B cells are pooled together and run through a FACSsystem. Only those B cells producing antibodies specific to the targetantigen will adhere to the antigen-expressing cells. Their adherenceshortens the distance between these two cells in the flow, compared tothe large separation between other individual cells, leading to a“bi-color event” detectable during their concurrent passing through thescanning laser beam. Thus, the B cells producing antibodies of interestcan be identified and then sorted into a different collection tube fromother non-specific B cells.

In another preferred embodiment, if the interaction between the B celland the corresponding antigen-expressing cell leads to certainmodification of cellular characteristics, e.g., depolarization,fluorescence resonance energy transfer (FRET), etc., more fluorescentlabels can be added to the cell capable of such modification. Thus, Bcells and antigen-expressing cells in contact will give out a“tri-color” event or an event containing even more than three colors atthe same time.

Alternatively, the identification and sorting of B cells in adherencedoes not require the existence of its fluorescent label. In oneembodiment, the cell-cell interaction leads to functional changes ineither cell. In another embodiment, these functional changes can be usedto identify and further separate B cells producing antibodiesspecifically binding to the target antigen. For example, the cell-cellinteraction may functionally block or activate receptor signaling ineither cell, leading to cellular changes, e.g., the Ca²⁺ efflux changes,etc., detectable by the FACS system. Thus, by monitoring thesedetectable functional changes, B cells of interest can also beidentified and separated. One particular embodiment of functionallyblocking or activating receptor signaling includes incubating B-cellswith cells that functionally express a GPCR (G protein-coupledreceptor). An agonist that signals through a GPCR can be added to themixture to induce GPDR mediated Ca²⁺ efflux from the endoplasmaticreticulum. In case an antibody presented on a B-cell would functionallyblock agonist signaling, Ca²⁺ efflux would consequently also be blockedby this cell-cell interaction. Ca²⁺ efflux can e.g. be quantitativelymeasured by flow-cytometry. Therefore, only B-cell/target cellconglomerates that either show increase or decrease in Ca²⁺ efflux wouldbe sorted.

Affinity Assays to Antibodies Produced by the Isolated B Cells

In certain embodiments, B-cells are cultivated under suitable conditionsso that antibodies are secreted into the culture medium. The producedantibodies are, for example, monoclonal antibodies. The cultivation mayinvolve the use of a helper cell line, such as a thymoma helper cellline (e.g. EL4-B5, see Zubler et al, 1985, J. Immunol., 134(6):3662-3668).

Optionally, additional affinity assays can be performed before furtherprocessing to evaluate the selectivity and the ability to compete withthe ligand of antibodies produced by the isolated B cells. These assaysinclude, but are not limited to, cell-based assays (e.g., cell ELISA(CELISA), which is a modified ELISA process in which entire cells areused for coating). As discussed in CA2350078, CELISA can be conducted asdescribed below in Examples. The validation step is performed to testthe generated antibodies for specific binding to the target, e.g. forexcluding antibodies which are directed against a protein beingexpressed on the cell surface other than the target protein.

Alternatively, the identified and isolated B cells of interest can bedirectly examined for antibody affinity and the B cells can be separatedfrom adhered antigen-expressing cells before the processing

Further Processing of Isolated B Cells for Antibody Production

The identified and isolated B cells, optionally tested by affinity assay(e.g., CELISA), can be further processed to produce immunobinders ofinterest. Traditional hybridoma technique, for example, can be used.This may involve steps such as purifying the immunobinders, elucidatingtheir amino acid sequence and/or nucleic acid sequence.

Alternatively, characterization of the binders is performed in theirscFv format. For this approach CDR sequences of binders expressed onsorted B-cells would be retrieved by RT-PCR from either the culturedsorted cells or from single cells directly. Combination of two pools ofpartially overlapping oligonucleotides in which one oligonucleotide poolis coding for the CDRs and a second pool encodes the framework regionsof a suitable scFv scaffold would allow for generation of a humanizedscFv in a one-step PCR procedure. HT sequencing, cloning and productionwould allow to perform clone selection based on the performance of thepurified humanized scFv, instead of characterizing secreted IgG in thecell culture supernatant. A scFv scaffold suitable to accept CDRs fromany rabbit antibody has been identified and characterized (“rabbitized”human FW or rabbit acceptor RabTor; see WO09/155726, which is herebyincorporated by reference in its entirety). The proof of concept for avariety of CDRs that in some cases even contain the rabbit specificinter-CDR disulfide bonds has been shown.

A general description of making rabbitized antibody is described below.

Grafting of Immunobinders

The antigen binding regions or CDRs of immunobinders identified usingthe methods of the invention can be grafted into acceptor antibodyframeworks. Such grafting can, for example, reduce the immunogenicity ofthe immunobinder or improve its functional properties, e.g., improvethermodynamic stability.

General methods for grafting CDRs into human acceptor frameworks havebeen disclosed by Winter in U.S. Pat. No. 5,225,539, which is herebyincorporated by reference in its entirety.

Specific strategies for grafting CDRs from rabbit monoclonal antibodiesare disclosed in U.S. Provisional Patent Application Nos. 61/075,697,and 61/155,041, which are hereby incorporated by reference in theirentirety. These strategies are related to that of Winter but diverge inthat the acceptor antibody frameworks are particularly suitable asuniversal acceptor for all human or non-human donor antibodies. Inparticular, the human single-chain framework FW1.4 (a combination of SEQID NO: 1 (named a43 in WO03/097697) and SEQ ID NO: 2 (named KI27 inWO03/097697)) has been shown to be highly compatible with theantigen-binding sites of rabbit antibodies. Therefore, the FW1.4represents a suitable scaffold to construct stable humanized scFvantibody fragments derived from grafting of rabbit loops.

Moreover, it was found that FW1.4 could be optimized by substituting 5or 6 residue positions in the heavy chain of FW1.4 and/or bysubstituting 1 position in the light chain of FW1.4. Thereby, it wassurprisingly found that loop conformation of a large variety of rabbitCDRs in the VH could be fully maintained, largely independent of thesequence of the donor framework. Said 5 or 6 residues in the heavy chainas well as the 1 position in the light chain of FW1.4 are conserved inrabbit antibodies. The consensus residue for the 5 or 6 positions in theheavy chain, as well as the one position in the light chain, was deducedfrom the rabbit repertoire and introduced into the sequence of the humanacceptor framework. As a result, the modified framework 1.4 (referred totherein as rFW1.4) is compatible with virtually any rabbit CDR. Contraryto the rabbit wild type single chains, rFW1.4 containing differentrabbit CDRs is well expressed and almost fully retains the affinity ofthe original donor rabbit antibodies.

Accordingly, exemplary immunobinder acceptor frameworks comprise

(i) a variable heavy chain framework having at least 70% identity,preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%identity, to SEQ ID No. 1; and/or

(ii) a variable light chain framework having at least 70% identity,preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%identity, to SEQ ID No. 2.

In a preferred embodiment, the variable light chain comprises Threonine(T) at position 87 (AHo numbering).

In a preferred embodiment, said immunobinder acceptor frameworkcomprises

(i) a variable heavy chain framework selected from the group consistingof SEQ ID No. 1, SEQ ID No. 4 and SEQ ID No. 6; and/or

(ii) a variable light chain framework of SEQ ID No. 2 or SEQ ID No. 9.

In a preferred embodiment, the variable heavy chain framework is linkedto a variable light chain framework via a linker. The linker may be anysuitable linker, for example a linker comprising 1 to 4 repeats of thesequence GGGGS (SEQ ID No. 10), preferably a (GGGGS)₄ peptide (SEQ IDNo. 8), or a linker as disclosed in Alfthan et al. (1995) Protein Eng.8:725-731.

In a most preferred embodiment, the immunobinder acceptor framework is asequence having at least 70%, 75%, 80%, 85%, 90%, more preferably atleast 95% identity, to SEQ ID. No. 3. More preferably, the immunobinderacceptor framework comprises or is SEQ ID. No. 3.

In another preferred embodiment, the immunobinder acceptor framework isa sequence having at least 70%, 75%, 80%, 85%, 90% more preferably atleast 95% identity, to SEQ ID No. 5. More preferably, the immunobinderacceptor framework comprises or is SEQ ID No. 5.

In another preferred embodiment, the immunobinder acceptor framework isa sequence having at least 70%, 75%, 80%, 85%, 90%, more preferably atleast 95% identity, to SEQ ID No. 7. More preferably, the immunobinderacceptor framework comprises or is SEQ ID No. 7.

Furthermore, an exemplary variable heavy chain framework of SEQ ID No. 1may be employed, further comprising one or more amino acid residues thatgenerally support conformation of CDRs derived from a rabbitimmunobinder. In particular, said residues are present at one or moreamino acid positions selected from the group consisting of 24H, 25H,56H, 82H, 84H, 89H and 108H (AHo numbering). These positions are provento affect CDR conformation and are therefore contemplated for mutationto accommodate donor CDRs. Preferably, said one or more residues areselected from the group consisting of: Threonine (T) at position 24,Valine (V) at position 25 Glycine or Alanine (G/A) at position 56,Lysine (K) at position 82, Threonine (T) at position 84, Valine (V) atposition 89 and Arginine (R) at position 108 (AHo numbering).

In a preferred embodiment, said variable heavy chain framework is orcomprises SEQ ID No. 4 or SEQ ID No. 6. Both variable heavy chainframeworks may for example be combined with any suitable light chainframework.

The sequences disclosed above are the following (X residues are CDRinsertion sites):

SEQ ID NO. 1: variable heavy chain framework of FW1.4 (a43)EVQLVESGGGLVQPGGSLRLSCAAS(X)_(n = 1-50) WVRQAPGKGLEWVS(X)_(n = 1-50)RFTISRDNSKNTLYLQMNSLRALDTAVYYCAK(X)_(n = 1-50) WGQGTLVTVSS SEQ ID NO. 2:variable light chain framework of FW1.4 (K127)LIVMTQSPSTLSASVGDRVIITC(X)_(n = 1-50) WYQQKPGKAPKLLIY(X)_(n = 1-50)GVPSRFSGSGSGALFTLTISSLQPDDFATYYC(X)_(n = 1-50) FGQGTKILTVLGSEQ ID NO. 3: framework of FW1.4 LIVMTQSPSTLSASVGDRVIITC(X)_(n = 1-50)WYQQKPGKAPKLLIY(X)_(n = 1-50)GVPSRFSGSGSGALFTLTISSLQPDDFATYYC(X)_(n = 1-50)FGQGTKILTVLGGGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAAS(X)_(n = 1-50) WVRQAPGKGLLWVS(X)_(n = 1-50)RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK(X)_(n = 1-50) WGQGTLVTVSS SEQ ID NO. 4:variable heavy chain framework of rFW1.4EVQLVESGGGLVQPGGSLRLSCTAS(X)_(n = 1-50) WVRQAPGKGLLWVG(X)_(n = 1-50)RFTISRDTSKNTVYLQMNSLRALDTAVYYCAR(X)_(n = 1-50) WGQGTLVTYSS SEQ ID NO. 5:framework of rFW1.4 EIVMTQSPSTLSASVGDRVIITC(X)_(n = 1-50)WYQQKPGKAPKLLIY(X)_(n = 1-50)GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)_(n = 1-50)FGQGTKILTVLGGGGGSGGGGSGGGGSGGGGSLVQLVLSGGGLVQPGGSL RLSCTAS(X)_(n = 1-50)WVRQAPGKGLLWVG(X)_(n = 1-50)RFTISRDTSKNTVYLQMNSLRALDTAVYYCAR(X)_(n = 1-50) WGQGTLVTVSS SEQ ID NO. 6:variable heavy chain framework of rFW1.4(V2)EVQLVLSGGGLVQPGGSLRLSCTVS(X)_(n = 1-50) WVRQAPGKGLLWVG(X)_(n = 1-50)RFTISKDTSKNTVYLQMNSLRALDTAVYYCAR(X)_(n = 1-50) WGQGTLVTVSS SEQ ID NO. 7:framework of rFW1.4(V2) EIVMTQSPSTLSASVGDRVIITC(X)_(n = 1-50)WYQQKPGKAPKLLIY(X)_(n = 1-50)GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)_(n = 1-50)FGQGTKILTVLGGGGGSGGGGSGGGGSGGGGS EVQLVLSGGGLVQPGGSLRLSCTVS(X)_(n = 1-50) WVRQAPGKGLLWVG(X)_(n = 1-50)RFTISKDTSKNTVYLQMNSLRALDTAVYYCAR(X)_(n = 1-50) WGQGTLVTVSS SEQ ID NO. 8:linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO. 9:substituted variable light chain framework of FW1.4EIVMTQSPSTLSASVGDRVIITC(X)_(n = 1-50) WYQQKPGKAPKLLIY(X)_(n = 1-50)GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)_(n = 1-50) FGQGTKILTVLG

Thus, unlike the general method of Winter, the framework sequence usedfor the humanization methods of the invention is not necessarily theframework sequence which exhibits the greatest sequence similarity tothe sequence of the non-human (e.g., rabbit) antibody from which thedonor CDRs are derived. In addition, framework residue grafting from thedonor sequence to support CDR conformation is not required.

The acceptor antibody frameworks can also comprise one or more of thestability enhancing mutations described in U.S. Provisional ApplicationSer. No. 61/075,692, which is hereby incorporated by reference in itsentirety. Exemplary solubility enhancing substitutions in the heavychain framework are found at positions 12, 103 and 144 (AHo numbering).More preferably, the heavy chain framework comprises (a) Serine (S) atposition 12; (b) Serine (S) or Threonine (T) at position 103 and/or (c)Serine (S) or Threonine (T) at position 144. Moreover, stabilityenhancing amino acids may be present at one or more positions 1, 3, 4,10, 47, 57, 91 and 103 of the variable light chain framework (AHonumbering). More preferably, the variable light chain frameworkcomprises glutamic acid (E) at position 1, valine (V) at position 3,leucine (L) at position 4, Serine (S) at position 10; Arginine (R) atposition 47, Serine (S) at position 57, phenylalanine (F) at position 91and/or Valine (V) at position 103.

EXAMPLES

Throughout the examples, the following materials and methods were usedunless otherwise stated.

Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, immunology (especially, e.g.,antibody technology), and standard techniques of polypeptidepreparation. See, e.g., Sambrook, Fritsch and Maniatis, MolecularCloning: Cold Spring Harbor Laboratory Press (1989); AntibodyEngineering Protocols (Methods in Molecular Biology), 510, Paul, S.,Humana Pr (1996); Antibody Engineering: A Practical Approach (PracticalApproach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: ALaboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); andCurrent Protocols in Molecular Biology, eds. Ausubel et al., John Wiley& Sons (1992).

Cell ELISA (CELISA)

The following example describes the use of CELISA to analyze cellsexpressing CXCR2:

CHO cells expressing CXCR2 can be seeded at a density of 50,000cells/well in 96-well half area plates. After overnight incubation at37° C., cells can be fixed with 1% formaldehyde in PBS for 30 minutes atroom temperature. Cell layers can be washed three times and non specificbinding sites can be blocked with cell culture medium for one hour atroom temperature. Next, after three washing steps, the supernatants canbe diluted 1:1 in culture medium and added to the wells. In threecontrol wells, a commercial rabbit anti-CXCR2 antibody can be spiked insupernatant. Supernatants can then be incubated on the cell layers forone and a half hours at room temperature. Finally, rabbit IgGs aredetected with a goat anti-rabbit IgG Fc antibody coupled to HRP. Uponaddition of a peroxidase substrate (Blue POD substrate from Roche), acolorimetric reaction would develop which could be stopped after 25minutes with 1M HCl. Absorbance can be measured at 450 nm.

Example 1 B Cell Screening System Using Soluble Antigens

A FACS (fluorescence-activated cell sorting)-based screening systemdescribed in this invention is exemplified here capable of selecting Bcells that bind to a target of interest, specifically, a solubleprotein, via their B cell receptors (BCR). In this Example, the targetwas the single-chain anti-VEGF antibody ESBA903 labeled with afluorescent dye (PE and PerCP). Lymphocyte suspension was prepared fromthe spleen of rabbits immunized with the recombinant target. Cells werethen incubated with PE and PerCP labeled scFv as well as with antibodiesspecific for IgG (APC-labeled) or IgM (FITC labeled). ESBA903-positiveB-cells that express IgG but not IgM on their surface were sorted andselected in 96-well plates (FIG. 2). As shown in panel A of FIG. 2,lymphocytes were gated according to forward and side scatter. Amongthem, IgG+ IgM− cells (probably memory B cells) were selected (panel B).Cells double-stained with scFv-PE and scFv-PerCP were expected to encodehigh affinity IgGs against the scFv (panel C). Cells showing thebrightest fluorescence were sorted in 96-well plates with sortingstatistics listed in panel D. By means of a thymoma helper cell line(EL4-B5: see Zubler et al, 1985, J. Immunol. 134(6):3662-3668), selectedB cells proliferated, differentiated into plasma cells and then secretedantibodies. The affinity of these IgG molecules for the target proteinwas verified by ELISA and Biacore measurements. Kinetic parameters aredepicted in Table 1 for seven selected clones. These clones, from a poolof ˜200 sorted cells, show high binding affinities in the low nanomolarto picomolar range. Finally, mRNAs for secreted IgG molecules wereisolated from 6 clones of interest and the CDRs were grafted on ESBATechsingle-chain framework RabTor, also termed Framework rFW1.4.

TABLE 1 Kinetic values for 7 B cell culture supernatants B-cell clone ka[Ms⁻¹] kd [s⁻¹] K_(D) [M] SG2 2.91E+06 2.95E−04 1.01E−10 SE11 3.63E+053.81E−04 1.05E−09 2E-03 8.34E+05 3.53E−04 4.23E−10 9E-03 8.66E+056.47E−04 7.47E−10 7D-03 3.97E+05 3.04E−04 7.65E−10 12B-02 1.08E+061.10E−04 1.01E−10 11G-02 5.48E+05 1.52E−04 2.78E−10 Sorting statisticsfor FIG. 2. Population #Events % Parent % Total All Events 100,000 ####100.0 Lymphocytes 86,585 86.6 86.6 Single Lymphocytes 1 86,013 99.3 86.0Single Lymphocytes 2 85,523 99.4 85.5 Memory B cells? 5,450 6.4 5.4Sorted Cells 16 0.3 0.0 903-binding cells 160 2.9 0.2

SELECTED LITERATURE

-   Zuber et al. Mutant EL-4 thymoma cells polyclonally activate murine    and human B cells via direct cell interaction. J Immunol. 1985;    134(6):3662-3668.    Transmembrane Targets

The screening system described above works efficiently when the targetis soluble, and when recombinant protein is available. However, sometargets of interest are multispan transmembrane proteins (e.g., GPCRsand ion channels). Traditional immunization with recombinant protein isin these cases inadvisable or impossible. Further, FACS selection of Bcells cannot be performed based on binding of purified, labeled proteinsif the antigen is an integral membrane protein. In order to addressthese issues the following improvements of the above mentioned procedurewere implemented.

1) Immunization with DNA Instead of Recombinant Protein:

DNA vaccination induces a rapid immune response against native antigen.Since no recombinant protein is needed, this technology is on one handvery cost-effective, on the other hand, and more importantly, thismethod allows for native expression of integral membrane complexesand/or multispan membrane proteins.

2) FACS Selection of B-Cells that Bind to Cells that Express an IntegralMembrane Target Protein:

Flow cytometry normally measures the fluorescence emitted by singlecells when they cross a laser beam. However, some researchers havealready used cytometers to investigate cell-cell interactions, forexample adhesion mediated by cadherins (Panorchan et al, 2006, J. CellScience, 119, 66-74; Leong et Hibma, 2005, J. Immunol. Methods, 302,116-124) or integrins (Gawaz et al, 1991, J. Clin. Invest, 88,1128-1134). However, such studies did not demonstrate whether suchcell-cell interactions will remain intact during the physical step ofcell sorting. Furthermore, it has never been shown that the binding of aB cell receptor to its target being present on the surface of anothercell will be strong enough to allow such physical sorting.

In order to select for B-cells that bind to transmembrane targets, cells(for example, CHO or HEK293 cells) can be transiently or preferentiallystably transfected with the target of choice, or cells that naturallyexpress the target of choice can be used. Such target cells are stainedwith an intracellular fluorescent dye (for example calcein) andincubated with the memory B lymphocytes of an immunized rabbit. Blymphocytes are stained with fluorescent antibodies binding to cellsurface specific markers. Thus, selection of bi-color “events”consisting in two cells adhering to each other through BCR-targetinteractions (see FIG. 1) can be achieved.

The further processing of these B cells is performed as described above,which leads to the production of monoclonal antibodies, for example inthe IgG or scFv format. To estimate the affinity of these antibodies forthe target, CELISA (ELISA, where coating step is performed with entirecells) is being performed. With this method, the selectivity and theability of antibodies to compete with the ligand can be evaluated.Finally, the CDRs of clones of interest will be cloned into ourrabbitized framework (RabTor) by gene synthesis with the oligo extensionmethod.

A read-out for B-cell sorting is not necessarily limited to cell-cellinteraction, but can also be used to select for the ability of thisinteraction to functionally block/activate receptor signaling. Forexample, B-cells can be incubated with cells that functionally express aGPCR. An agonist that signals through a GPCR can be added to the mixtureto induce GPCR mediated Ca2+ efflux from the endoplasmic reticulum. Incase an antibody presented on a B-cell functionally blocks agonistsignaling, Ca2+ efflux would consequently also be blocked by thiscell-cell interaction. Ca2+ efflux can be quantitatively measured byflow-cytometry. Therefore only B-cell/target cell conglomerates thatshow no Ca2+ efflux would be sorted.

Example 2 Detection of the Interaction Between Beads Coated withAnti-TNFα Antibody and CHO Cells Expressing Membrane-Bound TNFα

Before a B cell screening against a transmembrane protein is initiated,it has to be demonstrated that cell-cell interactions (and especiallyinteractions between BCR and transmembrane protein on target cell) canbe positively selected with a FACS. To determine whether the highpressure in the flow-cytometry stream breaks non covalent bindingbetween two cells, the following experiment was performed.

CHO cells stably transfected with membrane-bound TNFα (B-220 cells)(containing a mutant membrane-bound TNFα that contains a point mutationin the TACE cleavage site that prevents cleavage and shedding of TNFαligand; see, for example, Scallon et al. J Pharmacol Exp Ther 2002;301:418-26) were incubated with beads coated with a PE-labeled anti-TNFαantibody. In this set-up the beads mimic memory B cells. As negativecontrols, non-transfected CHO cells were used, as well as beads coatedwith an APC-labeled unrelated antibody (anti-CD19). After 2 hoursincubation at 4° C. with agitation, the cell-bead suspension wasanalyzed by FACS (using a 130 um nozzle). FIG. 3 shows that a specificbinding between anti-TNFα beads and TNFα-transfected CHO cells wasclearly detectable with FACS. Indeed, in this sample (upper panel) abouttwo thirds of the beads were bound to cells (585 bound against 267unbound). In contrast, in the control samples (middle and lower panels),almost no bead bound to CHO cells. Further, both bead populations(anti-TNFα-PE and anti-CD19-APC) were mixed together withTNFα-transfected CHO cells. FIG. 4 shows that about half of theanti-TNFα beads bound to CHO cells, whereas the vast majority of theanti-CD19 beads stayed unbound. The percentage of beads binding to thecell in each sample is detailed in Table 2. Thus, the demonstration ismade that the specific selection of single B cells that bind to anintegral membrane target protein through their B cell receptor waspossible using flow-cytometry.

TABLE 2 Percentage of beads bound to CHO cells in each sample Cells mAbon beads % bound beads Sample 1 CHO-TNFα (B220) anti-TNFα 68.0 Sample 2CHO-TNFα (B220) anti-CD19 0.9 Sample 3 CHO wt anti-TNFα 1.5 Sample 4CHO-TNFα (B220) anti-TNFα 47.0 anti-CD19 0.4

TABLE 2a Sorting statistics for upper panelof figure 3; binding of beadscoated with anti-TNFα antibodies to TNFα-transfected CHO cellsPopulation # Events % Parent % Total All Events 10,000 #### 100.0 P19,692 96.9 96.9 P3 585 6.0 5.9 P4 1 0.0 0.0 P2 267 2.7 2.7

TABLE 2b Sorting statistics for middle panel of figure 3; no binding ofbeads coated with anti-CD19 antibodies to TNFα-transfected CHO cellsPopulation # Events % Parent % Total All Events 10,000 #### 100.0 P19,399 94.0 94.0 P3 3 0.0 0.0 P4 6 0.1 0.1 P2 558 5.6 5.6

TABLE 2c Sorting statistics for lower panelof figure 3; no binding ofbeads coated with anti-TNFα antibodies to CHO wildtype cells Population# Events % Parent % Total All Events 10,000 #### 100.0 P1 9,001 90.090.0 P3 13 0.1 0.1 P4 7 0.1 0.1 P2 811 0.1 0.1

TABLE 2d Sorting statistics for figure 4 Population # Events % Parent %Total All Events 10,000 #### 100.0 P1 9,096 91.0 91.0 P3 401 4.4 4.0 P42 0.0 0.0 P2 856 8.6 8.6

Example 3 Detection of the Interaction Between B Cell Isolated from anAnti-TNFα Antibody Immunized Rabbit and CHO Cells ExpressingMembrane-Bound TNFα which were Saturated with Anti-TNFα Antibody

For the experiment depicted in FIG. 5, lymphocytes were isolated eitherfrom an anti-TNFα antibody (ESBA105, produced in-house) immunized rabbitspleen or from a non-immunized rabbit spleen. They were stained withanti-rabbit IgG-APC and anti-rabbit IgM-FITC (AbD serotec) andsubsequently pre-sorted in order to obtain pure populations of memory Bcells (IgG+/IgM−). In parallel, TNFα-expressing CHO cells (donated byDr. P Scheurich, Univ. of Stuttgart) were loaded with 1 ug/mLcalcein-red (Invitrogen), a cytoplasmic dye that fluorescently stainsliving cells. These cells were then washed once and incubated with (orwithout, for the negative control) 100 ug/mL of ESBA105, and finallywashed again 3× with PBS. Memory B cells were finally mixed at a ratioof about 1:10 with the CHO cells and incubated during 2 hours at 4° C.on a rotating plate (concentration: 3*10⁷ cells/mL). The followingsamples were prepared:

-   -   1) CHO-TNFα cells+ESBA105+ memory B cells of ESBA105 immunized        rabbit    -   2) CHO-TNFα cells+ESBA105+memory B cells of non-immunized rabbit    -   3) CHO-TNFα cells+memory B cells of ESBA105 immunized rabbit

After 2 hours incubation, the 3 samples were measured by FACS, using the70 um nozzle. According to the population hierarchy shown in Table 3a,5% of ESBA105 immunized cells bind to “ESBA105-coated” TNFα transgenicCHO cells. In comparison, only 0.5% non-immunized B cell bind tothese“ESBA105-coated” TNFα transgenic CHO cells (Table 3b), and 0.6% ofimmunized B cells bind in absence of ESBA105 on CHO cell surface (Table3c). These results give a show that a specific interaction between a BCR(B cell receptor) and a transmembrane target can be detected by FACS.

TABLE 3a Sorting statistics for upper panel of FIG. 5b Population#Events % Parent % Total All Events 50,000 #### 100.0 Living cells43,828 87.7 87.7 B cells 5,162 11.8 10.3 B cells sticking to CHO 78 1.50.2

TABLE 3b Sorting statistics for middle panel of FIG. 5b Population#Events % Parent % Total All Events 50,000 #### 100.0 Living cells43,834 87.7 87.7 B cells 4,290 9.8 8.6 B cells sticking to CHO 23 0.50.0

TABLE 3c Sorting statistics for lower panel of FIG. 5b Population#Events % Parent % Total All Events 50,000 #### 100.0 Living cells42,982 86.0 86.0 B cells 10,150 23.6 20.3 B cells sticking to CHO 65 0.60.1

Example 4 Detection of the Interaction Between B Cell Isolated from anESBA105 Immunized Rabbit and CHO Cells Expressing Membrane-Bound TNFαwhich were Saturated with ESBA105

In a further experiment, no pre-sorting of memory B cells was made. Theentire lymphocyte population was incubated with the stainedCHO-TNFα-ESBA105 cells. Transgenic CHO cells were prepared as describedabove. However, in order to prevent their proliferation in B cellculture medium after the sort in 96-well plates, the cells werecell-cycle arrested by a mitomycin C (M4287-2MG) treatment before thecalcein staining The lymphocytes of an ESBA105 immunized rabbit weremixed at a ratio of 3:1 with the stained CHO cells (concentration of thecell suspension: ≈3-10⁷ cells/mL), and incubated during 2 hours at 4° C.on a rotating plate. After this, the cell suspension was FACS analyzedand memory B cells binding to CHO-TNFα-ESBA105 were sorted according tothe gate depicted in FIG. 6, with 1, 10 or 100 cells/well as shown inTable 3. Sorted cells represented 5.5% of the memory B cell population,respectively 0.2% of total events.

Sorted cells were collected in 96-well plates and cultivated during 13days at 37° C. with 5% CO₂. After that, culture supernatants werecollected and tested in direct ELISA to check for the presence ofESBA105 binding IgGs. ELISA results (Table 3) show that ESBA105 specificantibodies could be detected in many wells, and also in wells wheresingle B cells were sorted. The Biacore (GE Healthcare) analysis (Table4) of these supernatants confirmed that these antibodies indeed bound toESBA105 target.

TABLE 3 ELISA analysis of B cell culture supernatant samples taken 13days after sorting. Day 13 0 B cell/well Positive control <> 1 2 3 4 5 67 8 9 10 11 12 A 0.0690 0.0680 0.0600 0.0470 0.0540 0.0700 0.0660 0.07500.0490 0.0690 2.7270 2.7020 B 0.0660 0.0630 2.7190 0.0460 0.0500 0.06400.0630 0.0770 2.7920 0.0580 2.8670 2.8880 C 0.0700 2.8380 0.0520 0.04700.0540 0.0690 2.9260 2.7160 0.0570 0.0690 2.9500 2.7730 D 0.0680 0.06300.0560 0.0490 0.0520 0.0640 0.0650 0.7840 0.4480 0.0620 3.0010 2.9250 E0.0750 0.0730 0.0630 0.0550 0.0630 0.0670 2.8550 0.0830 0.0580 1.98202.1250 2.8090 F 0.0680 0.0640 2.7090 0.0530 0.0580 0.0680 0.0750 2.56100.0610 0.3820 2.8150 2.8180 G 0.0820 0.0740 0.0660 1.7530 0.0700 0.07800.3980 0.0740 2.8920 1.8240 2.7910 2.7510 H 0.0730 0.0680 0.0620 0.05700.0610 0.0720 0.1710 1.9590 0.0610 2.7830 0.1800 2.8510 1 B cell/well 10B cells/well 100 B cells/well No B cells were sorted in raw A wells inorder to verify specificity of OD450 signals. In wells A11 and A12, apolyclonal rabbit anti-ESBA105 antibody (AK3A; 2 ug/mL) was spiked insupernatant as positive control. Wells where OD450 is significantlyhigher than background are highlighted in bold.

TABLE 4 kinetic values and concentrations determined by Biacore for theB cell culture supernatants. Fitted Chi2 Capture level CaptureApproximate Ka Kd % SE % SE KD Rmax (% of Bcell medium level B cell netcapture Concentration Protein (1/Ms) (1/s) (ka) (kd) (M) (RU) Rmax) withIgGs medium level (ug/ml) 19-01-B3 1.54E+06 2.83E−03 0.29 0.20 1.8E−0964.0566 0.30 772.65 554.36 218.30 0.273 19-01-C2 2.30E+09 3.23E+01 0.040.04 1.4E−08 99.0582 10.46 1069.06 546.00 523.06 1.576 19-01-F3 1.46E+062.31E−03 0.30 0.20 1.6E−09 77.848 0.37 803.86 537.13 266.72 0.34719-01-G4 3.40E+06 4.45E−03 2.86 2.29 1.3E−09 4.43221 1.43 578.01 548.5829.43 BQL 19-02-B4 8.18E+05 3.18E−03 0.22 0.15 3.9E−09 80.3549 0.22822.67 539.30 283.37 0.397 19-02-D3 1.01E+06 2.95E−03 0.54 0.36 2.9E−0923.7639 0.41 652.51 547.28 105.23 0.066 19-02-F2 2.61E+06 6.61E−04 1.530.59 2.5E−10 12.533 0.64 5214.04 514.43 9.61 BLQ Only supernatants whereone cell per well was sorted were measured. BLQ: below limit ofquantification

Example 5 Screening of Lymphocytes of Rabbits Immunized with CXCR2 Sorts27/29

Three rabbits were immunized with a CXCR2 expression vector. Afterseveral intradermal applications of CXCR2-cDNA, serum was taken andtested on CXCR2 transfected cells for presence of specific antibodies.Lymph node cells were then removed, frozen in five aliquots with each1.6×10⁷ cells and were stored in a liquid nitrogen tank.

An aliquot was thawed and stained with antibodies specific for IgG(APC-labeled) or IgM (FITC labeled). In parallel, CXCR2-expressing CHOcells were treated with mitomycin C, in order to prevent further growthwithout killing the cells, and loaded with 1 ug/mL calcein-red. Bothcell preparations were then mixed with a final cell concentration of 10⁷cells/ml, lymphocytes being twice as abundant as CXCR2-transfected CHOcells. After 2 hour incubation with gentle agitation at 4° C., cellsuspension was filtered through a 50-um filter and loaded on the FACS.Gating was performed as described in FIG. 6. One “event” (Memory B cellsbound to a CXCR2-transfected CHO cell) was sorted per well in a total of10×96-well plates (900 events in total). Sorted events represented 3.1%of the memory B cell population, respectively 0.035% of total cellamount in the sample.

Selected lymphocytes were cultivated during 21 days in a 37° C.incubator. Every 2-3 days, 100 uL of culture supernatant were collectedfrom the wells and replaced by fresh medium. During this culture time, Bcells proliferated, differentiated into plasma cells and secretedantibodies. In order to visualize which supernatants contained CXCR2specific antibodies, a CELISA was performed. For this, CHO cellsexpressing CXCR2 were seeded at a density of 50,000 cells/well in96-well half area plates. After overnight incubation at 37° C., cellswere fixed with 1% formaldehyde in PBS for 30 minutes at roomtemperature. Cell layers were then washed three times and non specificbinding sites were blocked with cell culture medium during one hour atroom temperature. Next, after three washing steps, the supernatants werediluted 1:1 in culture medium and added to the wells. In three controlwells, a commercial rabbit anti-CXCR2 antibody was spiked insupernatant. Supernatants were incubated on the cell layers during oneand a half hour at room temperature. Finally, rabbit IgGs were detectedwith a goat anti-rabbit IgG Fc antibody coupled to HRP. Upon addition ofa peroxidase substrate (Blue POD substrate from Roche), a colorimetricreaction developed which was stopped after 25 minutes with 1M HCl.Absorbance was measured at 450 nm.

This CELISA resulted in 1.8% of wells (16/900) displaying positivesignals. All positives were confirmed in a second CELISA. Supernatantswere also tested against other cell lines: CHO-K1 (wild type) to revealeventual unspecific binding clones, CHO-human CXCR1 and CHO-mouse CXCR2to demonstrate cross-activity against close-related receptor or speciescounterpart. Finally, supernatants were tested in a direct ELISA forbinding to a peptide consisting of the 48 CXCR2 N-terminal amino acids.Results are displayed in Table 5. All selected supernatants producedstrong OD₄₅₀ signals against human CXCR2 in CELISA. Some of them werealso slightly positive in the control experiment with the CHO wild typecells, meaning that they might bind in an unspecific way. None of theclones was cross-reactive with human CXCR1 or mouse CXCR2. Finally, someclones, but not all of them, bound to the CXCR2 N-terminal peptide,indicating a probable alternative binding site on CXCR2. Given theimpossibility of immobilizing entire cells on a Biacore chip, it iscurrently impossible to quantitatively measure the affinity of selectedantibodies for CXCR2 receptor. However, gathered data converge toindicate that antibodies specific to human CXCR2 were selected using thecell-cell interaction sorting system described above.

TABLE 5 Summary of CELISA results from anti-CXCR2 clones isolated duringsort 27 and 29 direct CELISA against CXCR2 rabbit IgG CELISA CELISAELISA N- Clone 1. Assay 2. Assay CHO wt. quantification CXCR1 mCXCR2term number OD₄₅₀ OD₄₅₀ OD₄₅₀ in ELISA (ng/mL) (OD₄₅₀) (OD₄₅₀) (OD₄₅₀)27-01-E3 3.3576 3.384 0.199 13501.9 0.1111 0.2210 2.3224 27-01-D9 3.17693.652 0.084 431.6 0.1100 0.1081 2.1632 27-01-H9 3.2068 3.707 0.3666439.8 0.3410 0.3180 0.0552 27-02-D3 2.1209 2.971 0.090 370.2 0.11900.1169 0.0507 27-03-H4 3.4092 3.490 0.373 904.3 0.4470 0.2562 2.714527-04-B3 2.7205 3.284 0.410 2896.1 0.4810 0.2724 0.0602 27-06-B5 0.44560.457 0.091 <5 ng/ml 0.1050 0.0980 0.05 27-06-A6 3.2461 3.507 0.423 12590.3640 0.2119 2.1627 27-07-B2 3.2434 3.390 0.140 455.9 0.1210 0.12162.4233 27-08-D5 3.1386 3.302 0.090 178.4 0.1060 0.0910 2.2873 27-08-G93.3705 3.302 0.100 427.6 0.1160 0.0881 2.4506 27-08-G11 3.2857 3.3800.125 2755 0.1660 0.1389 0.2955 27-09-A1 0.9547 1.926 0.103 261.1 0.11800.0996 0.0383 27-09-D1 3.2530 3.503 0.094 1576.5 0.1210 0.1238 0.050427-09-A5 3.2953 3.501 0.482 4502 0.4970 0.2491 2.5863 27-10-C3 0.64641.522 0.092 35.5 0.1030 0.1080 0.0513 29-01-H10 3.3345 3.3405 0.15585238.5 0.1810 0.1644 2.5374 29-02-C4 3.1456 3.3931 0.1219 2406.9 0.14900.1513 2.5126 29-02-H8 3.4441 3.3891 0.1178 4645.7 0.1260 0.1287 2.400329-02-C10 3.1259 3.4947 0.1128 861.3 0.1220 0.1074 1.9841 29-03-G112.5987 3.0270 0.1181 420.1 0.1110 0.0828 0.0501 29-04-F11 3.0250 3.18710.2768 16071.6 0.3160 0.1999 2.6047 29-05-E11 3.5481 3.4769 0.15312857.3 0.1950 0.1081 2.2094 29-06-H3 3.4308 3.4005 0.1254 8741.7 0.15300.1489 2.6543 29-06-D10 3.3152 3.4020 0.1316 1522.1 0.1210 0.1101 2.459829-07-H4 3.3693 3.4622 0.8502 16580.3 1.5030 0.7195 2.4458 29-08-E13.7283 3.4990 1.0667 10562.2 1.4780 0.5225 2.3015 29-08-G10 2.84292.5070 0.1107 40.4 0.1110 0.0955 1.8621 29-09-C4 1.1362 0.8767 0.1054 <5ng/ml 0.1090 0.0900 0.3539

EQUIVALENTS

Numerous modifications and alternative embodiments of the presentinvention will be apparent to those skilled in the art in view of theforegoing description. Accordingly, this description is to be construedas illustrative only and is for the purpose of teaching those skilled inthe art the best mode for carrying out the present invention. Details ofthe structure may vary substantially without departing from the spiritof the invention, and exclusive use of all modifications that comewithin the scope of the appended claims is reserved. It is intended thatthe present invention be limited only to the extent required by theappended claims and the applicable rules of law.

All literature and similar material cited in this application,including, patents, patent applications, articles, books, treatises,dissertations, web pages, figures and/or appendices, regardless of theformat of such literature and similar materials, are expresslyincorporated by reference in their entirety. In the event that one ormore of the incorporated literature and similar materials differs fromor contradicts this application, including defined terms, term usage,described techniques, or the like, this application controls.

1. A method for identifying an immunobinder that specifically binds to acell surface antigen of interest comprising: (a) providing a pluralityof immunobinder-expressing cells comprising a first sortable label; (b)providing a plurality of antigen-expressing cells comprising a secondsortable label, wherein the antigen of interest is displayed at thesurface of the antigen-expressing cell; (c) contacting theantigen-expressing cells with the immunobinder-expressing cells; and (d)separating from the plurality of immunobinder-expressing cells, one ormore immunobinder-expressing cells that can specifically bind to theantigen expressing cells using a cell sorter, wherein the presence ofthe first and second sortable label in a single cellular complex isindicative of the binding of an immunobinder-expressing cell to anantigen-expressing cell, thereby identifying an immunobinder that bindsto a antigen of interest.
 2. The method of claim 1, further comprisingclonally isolating the immunobinder-expressing cells obtained in step(d), optionally followed by clonal expansion of the clonally isolatedcells.
 3. The method of claim 2, further comprising obtaining theimmunobinder-encoding nucleic acid sequence from the isolatedimmunobinder-expressing cells.
 4. The method of claim 2, furthercomprising subjecting the isolated immunobinder-expressing cells to acell-based assay to functionally characterize the immunobinder.
 5. Themethod of claim 1, wherein the immunobinder is an antibody.
 6. Themethod of claim 5, wherein the antibody is a mouse, rabbit, chicken,camel, human, humanized, or chimeric antibody.
 7. The method of claim 6,wherein the antibody is a full length immunoglobulin, Fab, Dab, scFv, orNanobody.
 8. The method of claim 1, wherein the antigen of interest isexpressed from an exogenous gene.
 9. The method of claim 1, wherein theantigen of interest is a genetically engineered antigen expressed froman expression vector.
 10. The method of claim 1, wherein the antigen ofinterest is an integral membrane protein.
 11. The method of claim 10,wherein the integral membrane protein is a GPCR or an ion channel. 12.The method of claim 11, wherein the GPCR is CXCR2, CXCR1, CXCR3, CXCR4,CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2,CIC-4, CIC-5, CIC-7, CIC-Ka, CIC-Kb, Bestrophins, TMEM16A, GABAreceptor, glycin receptor, ABC transporters, NAV1.1, NAV1.2, NAV1.3,NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9, sphingosin-1-phosphatereceptor (S1P1R) or NMDA channel.
 13. The method of claim 1, wherein thefirst or second sortable label is a fluorescent label.
 14. The method ofclaim 13, wherein the fluorescent label is a fluorescent protein, anantibody/fluor conjugate, or a fluorescent cellular label.
 15. Themethod of claim 1, wherein the antigen-expressing cells are yeast cells,yeast spheroblasts or mammalian cells.
 16. The method of claim 15,wherein the antigen-expressing cells are human cells.
 17. The method ofclaim 1, wherein the immunobinder-expressing cells are yeast ormammalian cells.
 18. The method of claim 1, wherein theimmunobinder-expressing cells are B-cells.
 19. The method of claim 18,wherein the B-cells are rabbit 13-cells.
 20. The method of claim 18,wherein the B-cells are isolated from an immunized animal.
 21. Themethod of claim 20, wherein the animal is immunized by DNA vaccination.22. The method of claim 1, wherein the immunobinder-expressing cellscomprise an immunobinder expressed from an expression vector.